JP2798531B2 - Purification method of somatotropin monomer - Google Patents

Abstract

A method for purifying and recovering biologically active somatotropin monomers from refold solution following the solubilization and naturation of refractile bodies of host cells produced by recombinant DNA methodology. The purification process is based on the discovery that somatotropin monomers and somatotropin oligomers having overlapping isoelectric points may nevertheless be separated by selective precipitation over a narrow pH range. Host cell residues including proteins, pyrogens and other impurities present in the refold solution are effectively removed in the process. The purified somatotropin monomers recovered from solution after removing precipitated solids are suitable for parenteral application to target animals without further purification.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to the purification of somatotropin proteins produced by recombinant DNA technology, and more particularly to the separation and removal of somatotropin oligomers, residual host cell proteins and other impurities by a selective precipitation process. And purification and recovery of somatotropin monomers.

BACKGROUND Recombinant DNA technology invention, to allow the expression of heterologous protein in host cells such as E. coli bacteria. In the case of the growth hormone somatotropin, this protein is sequestered in a refractor in the cytoplasm of the host cell. The refractor can be recovered from the host cell culture by destroying the cells to release the refractor and then collecting the refractor as a solid pellet by differential centrifugation. The refractor is solubilized in an aqueous solution of a suitable chaotropic agent such as urea or guanidine hydrochloride at an alkaline pH, generally in the range of 10-12. The solubilized protein is then regenerated by contact with a mild oxidizing agent to form intramolecular disulfide bonds,
The protein is refolded or restored to its biologically active, raw conformation. Recombinant D
Methods for solubilizing and regenerating somatotropin proteins produced by E. coli bacteria using NA technology are described in US Pat. Nos. 4,511,502 and 4,652,630.
No. These descriptions are incorporated herein by reference.

[0003] The refolding solution obtained from the regeneration process consists of somatotropin monomers, dimers and higher oligomers, which contain residues from host cells and other debris. Of these, the somatotropin monomer is the desired biologically active ingredient and must be recovered in a highly purified form suitable for administration to the target animal by injection.

[0004] Purification of proteins is a common problem in biotechnology, and several methods have been developed to achieve such purification. J. Bonner
Biotechnology , 4th by Jea et al.
Vol., Pp. 955-958 (November 1986), "Protein Purification:
The Right Step at the Rig
The literature review entitled "ht Time" lists ten common methods for protein purification, with ion exchange chromatography, affinity chromatography and gel filtration being the three most common methods. According to the report in this document, to purify the protein homogeneously, an average of four steps of purification was required, the total yield was 28%, and the purification coefficient was 6,380. Is generally reported to involve precipitation, as a first or second step after homogenization.The most universal strategy of purification is homogenization followed by precipitation, ion exchange chromatography, affinity separation, Finally, gel filtration was performed, and it was stated that the precipitation yielded an average purification factor of three times.

[0005] Biologically active products derived from microbial sources and intended for parenteral administration to animals do not induce adverse reactions such as allergic reactions, fever and other side effects upon administration. Contaminants such as pyrogens, raw microbial proteins and nucleic acids must be substantially removed. John M. Curling et al .: Ame
rican Biotechnology Labor
aty, pages 33-37 (1983), "Downs.
stream Processing of Fermen
Tation Products by Chroma
1950, as described in
Invasive purification techniques, such as the precipitation method that has been used for plasma fractionation since the decade, cannot achieve the high levels of purity required for products produced with recombinant DNA. As a high-separation and purification technique that can be used in genetic engineering, chromatography is considered to be the first method selected at both research and production levels. The term "chromatography" includes the specific methods of ion exchange, gel filtration, affinity chromatography, hydrophobic chromatography and isoelectric focusing. Isoelectric point chromatography is a technique that separates proteins according to their isoelectric point in a resin bed with a pH gradient created by titrating a chromatography gel with a specially prepared buffer.

[0006] Chromatography has been reported to be the first choice method for the industrial recovery of ultrapure human proteins and hormones from bacterial homogenates for therapeutic applications. Chromatographic purification is preferably preceded by an initial fractionation step such as protein precipitation or liquid-liquid partitioning to increase the concentration in the chromatographic feed stream and improve efficiency, e.g. Dunnill: Process Biochem
issue , pages 9-13 (October 1983), "Tr
ends in Downstream Processes
sing of Proteins and Enzym
es ".

[0007] Examples of the use of chromatography in the purification of prochymosin expressed from E. coli by DNA recombination techniques are described in M. Emtage et al .: Bioche
mystry, Vol. 80, p. 3671-3675 (19
June 1983), "Synthesis of Calf
Prochymosin (prorennin) in
Escherichia coli ", which also describes most E. coli proteins (about 90%).
%) Precipitate under acidic conditions (pH 6.3) and can be removed by centrifugation.

[0008] Protein separation by the precipitation method is described in Albert
t, L. Lehninger: Bi ochemistr
y , 2nd edition, The John Hopkins Sc
cool of Medicine (1975), "T
he Molecular Basis of Cell
The textbook states that the solubility of a protein in a solution varies significantly as a function of pH, ionic strength, temperature and the dielectric constant of the solvent, and that the protein has its isoelectric point. pH, the P at which the molecule has no net charge
h explains that it is the least soluble. Under these conditions, the proteins tend to coalesce and precipitate. Certain proteins are practically insoluble at their isoelectric point pH, and as described in this document, when dissolved heterologous proteins have different isoelectric point pH values, They can be separated from one another by isoelectric precipitation.

Purification by fractional precipitation based on pH adjustment
In addition, Biochemical Engineerin
g Fundamentals , Bailey & O
Ilis, 2nd edition, pp. 745-749 (1986). Proteins having other properties such as the same molecular weight but different isoelectric points (pI) tend to precipitate proteins having the closest pI at a given pH, suggesting a method for separating proteins. By varying the pH, fractions containing different proteins can be separated.

[0010] The solubility of the protein is affected by the salt concentration and the temperature of the solvent.
In some cases, the solubility can be improved by further lowering or increasing the solubility of the protein at a low temperature. Isoelectric precipitation of poorly soluble proteins can also be improved by the addition of solutes such as ethanol or acetone or organic polymers such as polyethylene glycol in an aqueous medium. These methods are described in Protein Purificatio
n: Principles and Practic
e , R.C. K. Scopes, 2nd edition, 41-47 (1
987). A typical example of the use of the salting out method in the precipitation fractionation process described in this paper is pH
One-step purification of phosphate glyceraldehyde dehydrogenase from rabbit muscle by precipitation in aqueous solution containing 7.5 or more 3.2M ammonium sulfate.

As will be apparent from the foregoing, precipitation fractionation is an old, well-known method for achieving some degree of separation and purification of proteins in solution, and subsequent processing and high resolution. The technique has been accepted as a rough separation method to increase the concentration in the feed to the final purification by chromatography, for example. Although the specific conditions under which precipitation fractionation is performed will affect the yield and purity of the resulting protein, precipitation methods are used in the final purification of proteins intended for parenteral administration by injection into target animals. Never before. Bovine somatotropin produced by recombinant DNA technology for use in meat and dairy cattle, and the corresponding porcine somatotropin for growing pigs, have generally been purified by conventional chromatographic methods. One example of this type of method applied to an impure protein stock solution is the reverse method on a macroporous acrylate ester copolymer resin support as described in US Pat. No. 4,612,326. There is phase purification followed by elution with an organic diluent in aqueous solution at a pH of about 7-9.

The following US patents are of interest for their disclosure of various protein purification methods.

US Pat. No. 3,239,418 describes a two-step precipitation process followed by an ion exchange method for purifying the pituitary growth hormone promoter.

US Pat. No. 4,054,557 describes purification of a growth promoting polypeptide by gel filtration.

US Pat. No. 4,462,323 describes a two-step desalting and pH precipitation process in which impurities are first removed by precipitation, followed by precipitation and recovery of the desired protein.

US Pat. No. 4,451,487 discloses a process for purifying a biologically active protein by adding a selective precipitant which promotes precipitation of the desired protein while leaving impurities in solution. Is described.

US Pat. No. 4,569,794 describes the purification of biologically active polypeptides and proteins by chromatographic techniques.

US Pat. No. 4,617,376 describes a process for purifying glucagon from glucagon salt cake by hydrophobic chromatography.

The prior art of chromatographic purification of proteins, including somatotropin proteins, is complex and expensive. The precipitation method is relatively simple and inexpensive to operate, but fails to achieve the yields and resolution required for products intended for parenteral use.

Therefore, an object of the present invention is to provide a recombinant DN
An object of the present invention is to provide a method for purifying a somatotropin protein produced by the technique A.

Still another object of the present invention is to provide a method for separating somatotropin monomers from somatotropin dimers and higher oligomers contained in a solution.

It is yet another object of the present invention to provide a method for isolating and recovering somatotropin monomers from a solution containing somatotropin monomers and oligomers along with residual host cell proteins and other contaminants.

Still another object of the present invention is to provide an economical precipitation fractionation method for recovering high-purity somatotropin from a refolding solution in a regeneration step in a recombinant DNA method for producing somatotropin. It is in.

[0024] These and other objects and advantages of the present invention will become apparent to those skilled in the art from the following description and examples.

SUMMARY Briefly , the present invention provides a method for selectively precipitating somatotropin dimers and higher oligomers from a refolding solution with residual host cell proteins and other contaminants while refolding a bioactive somatotropin monomer.
The desired monomer is left in solution to provide a method for isolation and recovery. Precipitation increases the pH of the refolding solution from the initial high level of pH used for the solubilization and regeneration steps to the endpoint pH, typically about 4.5.
Achieved by lowering to a range of ~ 6. At this pH, substantially all host cell proteins and contaminant residues precipitate, most of the somatotropin oligomers precipitate, and most of the somatotropin monomers remain in solution. Isoelectric point chromatography reveals that somatotropin monomers exist as various "isoforms" with slightly different surface charges, and these isoforms can be classified into three main groups with significantly different isoelectric points. Has been. Surprisingly, it has been found that the somatotropin monomer can be efficiently separated from the oligomer by precipitation, even though the isoelectric points of the somatotropin monomer and the oligomer are in an overlapping range. Furthermore, it was found that no significant separation of these groups occurred, even though the three major groups of isoforms of somatotropin monomer had significantly different isoelectric points.

After the precipitated solids have been removed from the solution by filtration, centrifugation or other suitable means, the supernatant containing the dissolved somatotropin monomer is diafiltered (di
The solution is concentrated using a filtration or other suitable means to remove the buffer and the solution is then dried to recover the purified somatotropin product. The purified product is substantially free of residual bacterial proteins and other contaminants, pyrogens that cause adverse tissue reactions, etc., and is further purified by chromatography or other means commonly used prior to the present invention. Instead, it is suitable to be directly administered to a target animal by injection or transplantation. Purified products may contain from less than 1% to about 5% or more somatotropin oligomers. Somatotropin oligomers are biologically inert, but do not produce adverse reactions when administered to target animals.

This method describes in particular N-methionyl bovine somatotropin (MBST), N-alanyl bovine somatotropin (ABST) and N-alanyl butosomatotropin (PST).

DESCRIPTION OF THE DRAWINGS FIG . 1 is a photograph of an isoelectric point chromatography gel showing a partially isolated MBST somatotropin protein.
And 4 illustrate three major somatotropin monomer groups, labeled A, B and C, and lane 5 shows the somatotropin dimer.

FIG. 2 is a solubility curve of MBST monomer, MBST oligomer and Escherichia coli-derived protein residue.

FIG. 3 shows the effect of the concentration of refolded urea on the yield in the precipitation purification process of ABST.

FIG. 4 illustrates the effect of refolding urea concentration on the yield of the ABST refolding process.

Description of the Invention For the purposes of the present invention, the following terms should be considered to have the definitions set forth below.

The term "somatotropin" is meant to include, but is not limited to, mammalian somatotropins, such as humans, sheep, pigs and bovine somatotropins, and other such somatotropins. In addition to the above-mentioned somatotropin proteins having naturally occurring sequences being suitable, the present invention is equally applicable to systems involving homologs and analogs of naturally occurring proteins having somatotropin-like biological activity. It is. Therefore, such proteins are also included in the present invention as long as they are equivalent for purification purposes.

A "heterologous" protein is a protein that is not normally produced by its host cell. Recombinant DNA technology has allowed the expression of relatively large amounts of heterologous proteins, such as somatotropin, from transformed host cells, such as E. coli. However, although not completely understood, these heterologous proteins are often sequestered within insoluble refractions in the cytoplasm of the host cell.

"Refractive body" means an inclusion body or cytoplasmic granule containing at least a part of the heterologous protein to be recovered. These granules are seen as bright spots under a phase contrast microscope.

"Host cell" means any suitable cell, including microbial cells such as bacterial and yeast cells, or animal and plant cells transformed to express a heterologous protein. Host cells contemplated in the present invention include:
A cell in which a heterologous somatotropin expressed by the cell is sequestered within the refractor. An example of a host cell is E. coli K12, W3110G strain [pBGH1], which has been transformed to express bovine or porcine somatotropin.

"Regeneration" refers to the folding and oxidation of a heterologous somatotropin protein into its native conformation to ensure its biological activity.

"Folding" refers to the reconstitution of the overall conformational shape of a protein so that it can be properly oxidized. Folding is achieved when the amino acid sequences of a protein freely interact and adopt its native secondary and tertiary structure.

"Oxidation" refers to the formation of intramolecular disulfide bonds in the folded protein to stabilize the native conformation and ensure biological activity.

The term "refolding solution" means a raw solution obtained as a result of folding and oxidation in the regeneration step.

The effectiveness of pH precipitation in separating somatotropin monomers from somatotropin oligomers is:
Inconsistent with the usual theory of protein separation based on isoelectric point (pI). Somatotropins produced by recombinant DNA methods include three predominant groups of monomeric isoforms that differ substantially in isoelectric point. By analytical isoelectric focusing (IEF), these monomer groups are separated into distinct protein bands, as exemplified for MBST in FIG. Illustrative analytical IEF
Was performed on a flat bed electrophoresis apparatus. The gel contains 1% agarose, 10% sorbitol, 6M urea, and 2.
It contained 5% pH 5-9 Ampholines and was cast on FMC GelBond to a thickness of 1 mm. Focusing was performed at 15 ° C. and 10 watts constant power for 135 minutes. The gel was fixed and the focused proteins were stained and detected. Lane 1 is a Pharmacia standard IEF
Protein, lane 2 is a high purity monomer preparation produced by chromatographic isolation, lanes 3 and 4
Is an MBST monomer purified by the method of the present invention, and lane 5 contains a highly purified and isolated MBST dimer. The MBST monomers in lanes 3 and 4 consist of three major isoform groups, labeled A, B and C.

Further separation and analysis revealed that 3
Species of somatotropin monomer isoforms include N-
It was revealed that methionyl bovine somatotropin (MBST), N-alanyl bovine somatotropin (ABST) and N-alanyl butamatotropin (PST) were common. The relative proportions of the three isoform groups and the approximate average of their isoelectric points are shown in Table 1 below for a representative sample of each somatotropin product.

[Table 1]

The somatotropin dimers and higher oligomers do not show a constant isoelectric point value, as shown by the monomers (A), (B) and (C) above, as exemplified by the dimer in lane 5 of FIG. ) Has been found to be uniformly distributed over the entire range of pIs recorded.

In the following description and examples, the purification method of the present invention is based on the solution obtained by solubilizing and regenerating a refractor in an aqueous urea solution described in US Pat. No. 4,652,630. While illustrated with respect to purification of the folding solution, it should be understood that the invention is not limited by the specific details of the method used to generate the refolding solution. For example, the urea used to solubilize the refractors can be replaced by other chaotropic agents such as guanidine hydrochloride, sodium thiocyanate and various surfactants, or very high concentrations of sodium hydroxide.
The regeneration step can also be performed at a lower pH value and / or lower chaotropic agent concentration than used for the solubilization step.

In the case of the solubilization and regeneration method described in US Pat. No. 4,652,630, the components of a typical refolding solution are generally about 30-60% somatotropin monomer, about 10-30% somatotropin. Dimers and higher oligomers, as well as about 20-50% residues from E. coli bacteria, including but not limited to proteins, membrane fragments, plastids, endotoxins, pyrogens and nucleic acids. In addition, the refolding solution may contain urea at a concentration of about 1.5-6 M, depending on the type of somatotropin being oxidized. As disclosed in U.S. Pat. No. 4,652,630, a concentration of about 4-5M urea is preferred for bovine somatotropin regeneration, while a concentration of about 2.5-3.5M for porcine somatotropin is preferred. .

In a preferred embodiment of the present invention, the refolding solution obtained from the regeneration step is readjusted if necessary before proceeding with the precipitation fractionation method of the present invention. In particular, if the solution contains a sufficient amount of a chaotropic agent, such as urea or guanidine hydrochloride, to interfere with the precipitation process, the concentration of this agent must be reduced to acceptable levels. In the case of urea, concentrations above 3M have been shown to increase the solubility of somatotropin and hinder the precipitation process. Thus, if the refolding solution contains more than 3M urea, it is reduced to less than 3M, preferably 1M or less, by diafiltration or dilution. If the refolding solution contains a chaotropic agent other than urea, such as guanidine hydrochloride, measure the minimum concentration allowed for effective precipitation fractionation and adjust the concentration as necessary. There must be.

As described in US Pat. No. 4,652,630, the regeneration of solubilized somatotropin may be carried out at a low temperature, such as 4 ° C., to delay the degradation of urea, In the precipitation fractionation method of the present invention, the temperature is not particularly limited, and the refolding solution may be left to warm to room temperature, if desired. It is also true that somewhat higher temperatures in the range of 20-25 ° C. result in slightly better oligomer separation.

In one embodiment of the precipitation method of the present invention,
The pH of the refolding solution is slowly lowered from the high level used in the regeneration step, usually pH 10 or higher, by gradually adding an acid such as dilute acetic acid or other suitable organic or inorganic acid. In this case, mix well,
Avoid high acid concentration pockets. This is p
It will alter the H equilibrium and have a local effect on the solubility of the protein. The acid is added until a predetermined optimal pH endpoint value is reached. An optimal pH endpoint must be determined for each of the protein systems. This value is
The relative proportion of somatotropin monomers and oligomers,
It depends on the amount of bacterial proteins and contaminants present, and variables that affect the solubility of the protein, such as the presence or absence of various chaotropic agents, solvents, salts or solutes. Optimal pH endpoints generally range from 4.5 to 6, with virtually all bacterial residues and associated contaminants precipitating and most somatotropin oligomers precipitating in solution The amount does not exceed the highest level allowed for the purity of the final product, and most of the somatotropin monomer remains in solution and the amount precipitated is identified as a pH that does not exceed the maximum level in terms of acceptable yield. You.

In general, the yield of the process is inversely related to the purity of the final product. Since the high purity of the final somatotropin monomer product, which reflects the low level of oligomer, is obtained at the expense of yield, the maximum acceptable level of oligomer is set from an economic point of view. The somatotropin oligomers act as inert diluents in the final product, reducing the effective concentration of the active monomer component and increasing it accordingly requires an increase in the dose level administered to the target animal. It is believed that the oligomer does not have negative activity or cause undesirable side effects.

The actual relationship between yield and purity is, to some extent,
It depends on the upstream conditions used during the regeneration step and the quality of the feedstock processed in the precipitation fractionation step. In the refolding solution obtained from solubilization and regeneration of the refractor in urea as described above, the maximum allowable level of somatotropin oligomer in the preferred purified somatotropin product is 5% by weight, most preferably about 2.5%. Or less. The minimum acceptable yield of somatotropin monomer has been established at 50%, preferably at least 65%. The optimal pH endpoint for obtaining these results is
Although an individual determination must be made for each precipitation purification system, MBST refolding solutions generally range from 4.5 to 6, and most usually about 5.0 when treated as follows. It is.

The range of optimal pH endpoint values for a given system is based on the solubility data for the protein components of a typical MBST refolding solution obtained from the urea system described above, as exemplified in FIG. provide,
It can be predicted based on the protein solubility curve. As illustrated in this figure, residual proteins and impurities from E. coli precipitate substantially completely in the pH range of about 4.5-7.2. The amount of somatotropin oligomer in solution relative to total MBST is less than 1% in the pH range ranging from about 5.5 to 7.2, but increases rapidly as the pH falls below 4.5. Somatotropin monomers have minimal solubility at about pH 6.5, and this solubility increases rapidly as the pH is reduced to levels below 6. The optimal pH endpoint for this particular system is about 5.0 ±
At this pH, the bacterial residue is substantially insoluble and the solubility of the somatotropin oligomer is about 2.5%
So that the purity of the final MBST product is about 9
It becomes 7.5%. Also, more than 80% of the somatotropin monomer initially present in the system remains in solution, giving acceptable yields.

The optimum end point pH and the yield and purity of the product obtained from this precipitation process are influenced by the conditions used for the regeneration step, in particular the refolding pH, protein concentration and urea concentration. The data shown in Table 2 shows the optimal end point pH and 2
5 illustrates the effect on the precipitation yield in the case of producing ABST containing 0.1% oligomers.

[Table 2]

The effect of the somatotropin protein concentration during the regeneration step on the precipitation yield of 2% oligomer-containing ABST and 2% oligomer-containing PST is illustrated by the data in Table 3.

[Table 3]

This effect of refolding pH and protein concentration on subsequent purification of somatotropin monomers results in reduced precipitation yields due to the high levels of oligomers and / or other impurities produced under refolding conditions. It is considered something. Therefore, an important feedstock quality to consider in the process of the present invention, the solubilization and regeneration steps maximize monomer yield and reduce the amount of oligomers and other undesirable components removed in the process of the present invention. It should be operated in a manner consistent with other considerations of the process, such as minimizing concentration.

In the case of MBST, the effect of the refolding urea concentration on the precipitation yield is illustrated in FIG. In this case, the yield of the final purification step appears to increase as the concentration of urea used in the regeneration step increases from 2.5M to 5.5M. However, as further illustrated in FIG. 4, the product yield in the regeneration step is maximum at 4.5M urea and falls off rapidly as the concentration of urea increases to 5.5M. That is, the efficiency of the whole process is
A function of the individual yields at each step of the process,
The urea concentration during regeneration must be chosen to maximize the overall process yield. For the systems illustrated in FIGS. 3 and 4, the optimal urea concentration in the regeneration step is about 4.5M, which seems to maximize the overall product yield.

The precipitation purification method of the present invention will now be illustrated by the following examples aimed at the purification of N-alanyl bovine somatotropin and N-alanyl butamatotropin. In the examples, all percentages are weight percentages.

Example 1 A refractor expressed in E. coli and containing N-alanyl bovine somatotropin (ABST) was produced and isolated using known techniques. Sufficient amount of refractor is adjusted to pH 11.2
5. When solubilized in a 4.5 M aqueous urea solution at a temperature of 4 ° C., an ABST solution having a concentration of about 3.4 g / l was obtained. The refractor dissolved quickly and the resulting solution was regenerated with stirring at 4 ° C. for several days or until at least about 95% of the ABST was oxidized and refolded into its native form. HPL of oxidized and refolded solution
C analysis showed an ABST yield of about 73%.

The refolded solution was Mil
lipore (MilliporeCooperati
on, 80 Ashby Rd. , Bedford, MA
01730) PLGC, nominal molecular weight limit 10,000
The solution was concentrated to 600 ml by diafiltration using a membrane and then replaced with 5 volumes of cold deionized water. The resulting solution was an aqueous solution of about pH 10 containing about 25 g / l ABST monomer, 8 g / l ABST oligomer, 27 g / l E. coli residue, and less than 0.2 M urea.

The solution thus prepared according to conventional and known procedures was then purified by the method of the present invention to recover the ABST monomer as follows. 200 ml of prepared solution
Was warmed to room temperature, 5% acetic acid was gradually added, and the pH of the solution was lowered to 4.8 over 3 hours to form a precipitate. The solution was maintained at pH 4.8 and stirred for 1 hour, then centrifuged to remove the precipitate. According to the analysis, the protein dissolved in the clear supernatant was measured by ELISA for 2.
It contained 3% ABST oligomer and less than 0.1% E. coli residues, and the purity of the ABST monomer was 97%. 83% yield of ABST monomer during purification process
It was calculated. The purity of the ABST monomer was confirmed by SDS polyacrylamide gel electrophoresis.

Example 2 A refractor expressed in E. coli and containing N-alanyl butosomatotropin was prepared and isolated by known techniques. A sufficient amount of the refractor is solubilized in 2 liters of a 3M aqueous urea solution at a pH of 11.25 and a temperature of 4 ° C. to give a P concentration of about 3 g / l
An ST solution was obtained. The refractor dissolved rapidly and the resulting solution was regenerated by stirring at 4 ° C. for several days to oxidize the PST and refold to its native form. HPL of oxidation and refolding solution
In C analysis, the yield of PST monomer was about 70%.

The refolding solution was Millip
ore PLGL, concentrated to 200 ml by diafiltration using a membrane with a nominal molecular weight limit of 10,000, followed by 5 ml.
Replaced with a volume of cold deionized water. The obtained solution is prepared by adding about 20 g / l of PST monomer, 7
g / l PST oligomer, 15 g / l E. coli residue and less than 0.2 M urea.

The solution thus prepared according to conventional and known procedures was then purified by the method of the present invention as follows to recover the PST monomer. Prepared solution 1
8.75 g of Celite 577 filter adjuvant are added to 75 ml in advance, warmed to room temperature, and the pH of the solution is lowered to 4.7 by slowly adding 5% acetic acid over 1.75 hours to form a precipitate. I let it. PH 4.7 for 1 hour while stirring the solution
, And centrifuged to remove the precipitate. Analysis revealed that the protein dissolved in the clear supernatant was 2.6% PST oligomer and 0.01% as determined by ELISA.
Less than Escherichia coli residue and the purity of PST monomer is 9
It was found to be 7 +%. PST in the purification process
The monomer yield was calculated to be 84.5%. The purity of the PST monomer was confirmed by SDS polyacrylamide gel electrophoresis.

In a variation of the direct precipitation method described above, the pH of the refolding solution is first reduced to a level at which substantially all solubilized somatotropin monomer remains in solution, and then the pH is increased to an optimal endpoint. Thereby, the optimum end point pH can be approached from the acid side.
According to the MBST solubility curve of FIG. 2, acetic acid was added to the cold refolding solution at pH 10 to adjust the pH to about 4.5.
It can also be reduced to less than. At this pH level,
All solubilized proteins remain substantially dissolved in the aqueous urea solution, and the protein precipitated during the addition of acetic acid is pH
Is further reduced to 4.5 or less, and it can be seen that it is redissolved. The concentration of urea or other chaotropic agent present in the solution can then be reduced, if necessary, by diafiltration or other suitable means. next,
The pH is adjusted by the addition of NaOH or other suitable base to the precipitation of substantially all oligomers and E. coli proteins, while the optimal pH endpoint indicated by the high yield of MBST monomer remaining in solution. Adjust upwards until achieved. The precipitate is removed and purified MB
The ST monomer is recovered from the solution as described above.

The quality and efficiency of the feedstock of the precipitation and purification method of the present invention may, in some cases, be controlled by additional washing of the refractor prior to the somatotropin protein solubilization and regeneration steps to reduce contaminating cell debris. It can be improved by removal. In some cases, washing with water or an aqueous solution containing a surfactant, solvent or NaOH helps to remove many soluble impurities, and reduces overall process yield and purity of the final somatotropin monomer product. Significant improvement.

Alternative Embodiment- Two-Step Precipitation In another embodiment of the invention, the pH of the initial refolding solution is increased above the optimal pH endpoint by leaving at least about 80% of the somatotropin monomer in solution. However, up to about 30% of the somatotropin oligomer remains in solution, reducing to a point where up to about 10% of the small amount of residual host cell protein remains in solution. The precipitate formed consists mainly of bacterial debris and contains significant amounts of somatotropin oligomers. When this is removed from the system by filtration or centrifugation, a solution rich in somatotropin monomer remains, containing less than about 20% somatotropin oligomers and less than about 5% bacterial debris, based on total dissolved protein. Is preferred. The pH of the concentrated solution is precipitated with the addition of NaOH or other suitable base along with substantially all of the host cell debris where most of the remaining somatotropin oligomers are present, resulting in a highly purified solution of the somatotropin monomer. Lower to the remaining optimal endpoint value. The advantage of this two-step precipitation process lies in the fact that the concentrated feedstock used for the second precipitation gives high purity somatotropin monomer.

The two-step precipitation method can be more easily understood with reference to FIG. As described above in connection with the single-step precipitation method, the optimal pH endpoint for the separation and recovery of MBST monomer in this system is about 5.0 ± 0.2, at which pH substantially all Bacterial residue precipitates,
The concentration of somatotropin oligomer remaining in the solution is about 2.
5%. By further reducing the pH to about 4.3, substantially all of the precipitated somatotropin monomer was redissolved, while about 30% of the somatotropin oligomer was redissolved and the concentration in the solution was about It rises to 10% by weight. However, the bacterial residue remains substantially insoluble at this pH. Separating the precipitate at this point removes substantially all bacterial residues and about 70% of the somatotropin oligomers originally present in the system. The pH of the remaining solution was then increased to the optimal endpoint of the second precipitation, whereupon most of the remaining somatotropin oligomers and any residual bacterial proteins still remaining in the solution also precipitated out. Further fractionation occurs. The optimal endpoint value for the second precipitation need not be the same as for the single precipitation. Generally, the optimal endpoint pH of the second precipitate is between 5.5 and 8.5 for bovine somatotropin.
In the case of porcine somatotropin would be 5.5-7.5.

The two-step precipitation method is illustrated by the following example.

Example 3 200 ml of MBST refolding solution at pH 11 in 4.5 M aqueous urea was added to about 50 l of 1 mM NaOH.
Dialysis at 4 ° C. for 16 hours to reduce the urea concentration to less than 0.2M. This solution was added to an Amicon stirred cell (Amicon Div) equipped with a YM10 membrane.
issue, W.M. R. Grace & Co. , Danv
ers, MA01923). At this point, the solution contains about 7.0 g / l MBST monomer,
3.4 g / l MBST oligomer and 2.1 g / l
It contained 1 E. coli residual protein. The solution was warmed to room temperature and the pH was reduced to 5.0 by the dropwise addition of dilute acetic acid.
At this point significant precipitation occurred. The solution was centrifuged to remove the precipitate. The clear supernatant was collected and NaOH was added to p.
H was adjusted to 6.0. One hour later, the sample was centrifuged to remove the second precipitate, and the MBST monomer in the clear supernatant was recovered. Table 4 shows the purity and yield in each step of this process.

[Table 4] The overall process yield is about 76%.

In MBST purification, the pH endpoint for the first precipitation is usually in the range of 4.0 to 5.5,
On the other hand, the pH endpoint for the second precipitation is usually 5.5-6.
5 range. In the case of ABST, the first pH endpoint is usually in the range of 4.0 to 5.5, while the second pH endpoint is usually in the range of 6.5 to 8.5. In the case of PST, the first pH endpoint is usually in the range of 4.0-5.0, while the second pH endpoint is usually in the range of 5.5-7.5. In all cases, the first pH endpoint is that most of the somatotropin oligomers and E. coli residues will precipitate, while at least about 80
%, Preferably 90-95% or more, is selected to remain in solution.

In the two-step precipitation process, the level of oligomers in the final product can be easily reduced to less than 2%, often less than 1%, and the final purity of the somatotropin monomer recovered is 99%. Beyond.

Although the foregoing description and examples have been directed to specific somatotropin protein systems, the methods of the present invention are generally applicable to the purification of MBST, ABST, PST and other somatotropin variants. It should be understood that Optimum pH endpoints and other process variables will vary depending on the particular conditions of a particular system, but they can be readily determined from protein solubility curves and few experiments. The relative solubility of the protein in solution is affected by temperature or the presence of various salts, solvents, solutes, etc., and increases the solubility difference using the above variables to enhance fractionation during the precipitation step Doing so is included within the scope of the present invention. Solvent modifiers that can be used to enhance fractionation include alcohols such as ethanol and isopropanol, nonionic surfactants such as Triton X-100 and Tween 80, polyethylene glycol, sugars, and various salts. Other elements of the method, such as acids or bases used for pH adjustment, can be selected by the practitioner, depending on personal preference. The pH can be lowered, for example, using acetic acid, phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid or citric acid, but the optimal pH endpoint may shift slightly upward or downward for different acids. pH is NaOH, KOH, N
Elevation can be achieved with H ₄ OH, tris (hydroxymethyl) aminomethane or ethanolamine, but again the optimal pH endpoint may be shifted by the particular base selected. All variables of such a method are within the scope of the present invention.

[Brief description of the drawings]

FIG. 1 is a photograph of an isoelectric point chromatography gel of partially purified MBST somatotropin. Lane 1: Pharmacia standard IEF protein Lane 2: High purity monomer sample Lanes 3 and 4: Monomer purified by the method of the present invention Lane 5: Purification Dimer.

FIG. 2 shows solubility curves of MBST monomers, MBST oligomers, and protein residues derived from Escherichia coli.

FIG. 3 is a graph showing the influence of the urea concentration on the yield in the precipitation purification process of ABST.

FIG. 4 is a graph showing the effect of urea concentration on the yield of the ABST refolding process.

Continuation of front page (56) References JP-A-61-195698 (JP, A) JOURNAL OF CLINICAL ENDOCRINOLOGY A ND METABOLISM (1973), VOL. 37, NO. 6, P. 860-866 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 21/00, 21/02 C12N 15/00 CA (STN)

Claims (35)

(57) [Claims] 1. An oxidized refolding solution comprising a somatotropin monomer and a somatotropin oligomer in an aqueous solution having a pH of 7 or more, and
The pH is lowered to 4.5-6.0 to selectively precipitate somatotropin oligomers from this solution, while most of the somatotropin monomer remains in solution, and c) the precipitate is removed to remove somatotropin remaining in solution. A method for separating a somatotropin protein, which recovers the protein. 2. The method according to claim 1, wherein the obtained refolding solution is composed mainly of somatotropin monomer and small part of somatotropin oligomer. 3. The method according to claim 2, wherein the somatotropin oligomer is present in an amount of 10 to 30% by weight of the total protein. 4. The method according to claim 3, wherein the amount of the somatotropin oligomer remaining in the solution after precipitation is less than 5% by weight of the total somatotropin protein remaining in the solution. 5. The obtained refolding solution has a pH of 1.
2. The method according to claim 1, wherein the method is present in zero or more aqueous solutions. 6. The method according to claim 1, further comprising the step of removing volatile substances from the recovered solution to obtain a dried somatotropin protein product. 7. The method according to claim 1, wherein the somatotropin protein is bovine or porcine somatotropin. 8. The method according to claim 7, wherein the somatotropin is N-methionyl or N-alanyl bovine somatotropin. 9. The method according to claim 7, wherein the somatotropin is N-alanylbutamatotropin. 10. A mixture of a protein consisting of a somatotropin monomer, a somatotropin oligomer and a bacterial residue in an aqueous solution having a pH of 7 or more, b) the pH of the solution
To 4.5 to 6.0 to selectively precipitate somatotropin oligomers and bacterial debris from this solution, while most somatotropin monomers remain in solution,
c) A method for separating and purifying a somatotropin protein, in which the precipitate is removed and the purified somatotropin protein remaining in the solution is recovered. 11. A solution of the protein in solution comprising recombinant DN
The method according to claim 10, which is obtained by regenerating a somatotropin protein produced from a host cell culture by the A technique. 12. The method according to claim 11, wherein the bacterial residue is derived from an E. coli host cell. 13. The method of claim 10, wherein a majority of the bacterial residue is selectively precipitated with the somatotropin oligomer. 14. The method of claim 10 wherein the resulting solution consists of at least 30% somatotropin monomer, 10-30% somatotropin oligomer, and 20-50% bacterial residue. 15. The method according to claim 14, wherein the protein remaining in the solution after precipitation consists of at least 95% somatotropin monomer, less than 5% somatotropin oligomer and less than 1% bacterial residue. 16. The method according to claim 10, further comprising the step of removing volatile substances from the solution remaining after precipitation to obtain a dried somatotropin protein product. 17. The method according to claim 10, wherein the aqueous solution contains a chaotropic agent. 18. The method according to claim 17, wherein the chaotropic agent is selected from the group consisting of urea, guanidine hydrochloride, sodium thiocyanate and a surfactant. 19. The method according to claim 17, wherein the chaotropic agent is urea, and the concentration of the urea in the precipitation step is less than 3M. 20. The method according to claim 19, wherein the obtained protein mixture is present in an aqueous solution having a pH of 10 or more. 21. The method according to claim 10, wherein the somatotropin protein is bovine or porcine somatotropin. 22. The somatotropin is N-methionyl or N-alanyl bovine somatotropin.
1). 23. The method according to claim 21, wherein the somatotropin is N-alanylbutamatotropin. 24. a) obtaining a mixture of somatotropin monomers and somatotropin oligomers in an aqueous solution with a bacterial residue at a pH of 7 or more, b) causing the pH of the solution to precipitate a substantial portion of the somatotropin oligomers and bacterial residues, Reducing the somatotropin monomer to a first value effective to remain in solution; c) removing the precipitate from the solution; d) selectively increasing the pH of the solution and further somatotropin oligomers and bacterial residues from the solution. Let
On the other hand, a method for separating and purifying a somatotropin monomer in which most of the somatotropin monomer is raised to a second endpoint value effective to remain in the solution, and e) the precipitate is removed to recover the purified somatotropin protein in the solution. 25. The somatotropin is bovine somatotropin, wherein the first pH value is between 4.0 and 5.5 and the second pH value is
The method according to claim 24, wherein the value is 5.5 to 8.5. 26. The method according to claim 25, wherein the somatotropin is N-methionyl bovine somatotropin, and the second pH value is 5.5 to 6.5. 27. The method according to claim 25, wherein the somatotropin is N-alanyl bovine somatotropin and the second pH value is 6.5 to 8.5. 28. The somatotropin is N-alanyl butamatotropin, wherein the first pH value is 4.0-5.0 and the second pH value is 5.5-7.5. The described method. 29. The method of claim 24, wherein at least 80% of the somatotropin monomer remains in solution at the first pH value. 30. The method of claim 24, wherein the level of somatotropin oligomers in the solution at the second endpoint pH is less than 2% by weight of total somatotropin protein in the solution. 31. a) obtaining a mixture of a somatotropin protein consisting of a somatotropin monomer and a somatotropin oligomer in an aqueous solution having a pH of 7 or more; b) maintaining the pH of the solution at 4. while substantially maintaining the monomer and oligomer in the solution. C) by precipitating the somatotropin oligomers from this solution, while keeping most of the somatotropin monomer in solution by raising the pH of the solution above 5.5, and then d) A method for separating somatotropin proteins, comprising removing a precipitate and recovering somatotropin proteins remaining in the solution. 32. The method according to claim 31, wherein the aqueous solution contains a chaotropic agent in an amount sufficient to substantially increase the solubility of the somatotropin protein. 33. The method according to claim 32, wherein the chaotropic agent is selected from the group consisting of urea, guanidine hydrochloride, sodium thiocyanate and a surfactant. 34. The method of claim 32, wherein the chaotropic agent is substantially removed from the aqueous solution prior to the selective precipitation of the somatotropin oligomer. 35. The method according to claim 31, wherein the somatotropin protein is bovine or porcine somatotropin.